Summary of Work: The goal of this project is to develop therapeutic radiopharmaceuticals based on targeting the decay of Auger electron emitting radioisotopes to specific sequences in DNA (genes), using triplex-forming oligonucleotides as delivery vehicles. In in vitro studies we have demonstrated that triplex-forming oligonucleotides (TFOs) are able to deliver Auger electron emitters to specific targets in cellular DNA in order to inactivate genes and/or kill the cells containing the target sequences. Decay of 1251 in TFOs results in strand breaks in both strands of the target DNA with an efficiency from 0.4 to 0.8 break/ decay. Higher efficiency can be achieved with radionuclide multiple labeling. Breaks are confined to the triplex target sequence, and 90% of the sequence-specific breaks are located within 10 bp of the decay site. Specificity of TFOs was shown to be high enough to specifically break genomic DNA in a target located in a single copy gene. A liposome delivery system has been developed to effectively deliver radiolabeled TFOs into the cell nucleus. Radiotoxicity of TFOs delivered into the cell nucleus as measured by clonogenic assay is 300 times less than that of DNA-incorporated 125IUdR. A new generation of chemically modified triplex-forming oligonucleotides with increased in vivo stability, permitting onestep labeling with Auger electron emitters, is being developed. We have also shown that the fine structure of DNA damage by decay of Auger electron emitter depends on local DNA conformation. Therefore, by analyzing the DNA damage one can obtain information on the structure of DNA in nucleoprotein complexes both in vitro and in vivo. Based on this principle, a new method of radioprobing of DNA-protein complexes has been demonstrated in several model systems. In addition, studies have been initiated to investigate the mechanisms of Auger electron-induced DNA strand break repair in human cells. The aim of these studies is to identify methods by which human repair processes may be manipulated to augment the radiotherapeutic effects of Auger electron-emitting TFOs.